During FY10, we focussed on three subprojects relating respectively to hepatitis B virus, bacteriophage HK97, and bacteriophage phi6.&#8232;&#8232; (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (capsids) and e-antigen (unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into shells of two different sizes, we obtained, in 1997, a cryo-EM density map in which we visualized the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. Our subsequent research helped delineate the path of the polypeptide chain. We went on to investigate the antigenic diversity of HBV by using cryo-EM to characterize the conformational epitopes of seven different monoclonal antibodies raised against capsids. In FY11, we pursued two lines of investigation. (i) Expression, purification, and co-crystallization of e-antigen (eAg). The- eAg polypeptide differs from that of core antigen (cAg) in having at its N-terminus an additional ten residues, a remnant of its propeptide, and in lacking the basic 34-residue C-terminal peptide of cAg. eAg and cAg are antigenically distinct but are cross-reactive, as 4 of 6 monoclonal antibodies tested in a recent study bound both antigens (2). One of the two exceptions is Mab e6, whose Fab forms a stable complex with eAg. We have now characterized this complex further. Crystals have been grown which diffract synchrotron radiation to 3.3 resolution. The diffraction data index according the triclinic space group P1 with unit cell parameters: a = 66.7, b = 75.8, c = 88.7. Structure determination is in progress. We also continued our program to analyze the epitopes of anti-cAg antibodies by cryo-EM and molecular modelling, and made progress in two directions. In previous studies, we identified the epitopes of seven murine monoclonal antibodies. Seeking to relate these observations to the immunological response of infected humans, we isolated anti-cAg antibodies from a patient, prepared Fabs, and analyzed their binding to capsids. The observed Fab-related density was highly differentiated and could be reproduced by modelling with just five Fabs whose locations match those of the murine monoclonals previously studied. These results validate the mouse as a model system. In a previous study (by others), the sera of two unrelated patients suffering from acute liver failure (fulminant hepatitis) were found to contain a common antibody specific for cAg, We have found that whereas most antibodies previously characterized bind around the tips of protruding spikes, the ALF-associated antibody binds tangentially to the side of the spike in a region in which epitopes have not been detected before. These results support the proposition that antibodies with particular specificities may correlate with different stages of disease progression. (2) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids, reflecting common evolutionary origins. In FY11, we focussed mainly on the following investigations. (2a) Thermally-induced Phase Transitions in Bacteriophage Capsids. The capsids of tailed phages assume multiple conformational states, depending on their state of maturation and DNA packaging. For example, the HK97 capsid passes through three intermediate states en route from the earliest precursor procapsid to the fully mature capsid. Switching between conformations represents a phase transition of the capsid surface lattice. Our previous calorimetric studies have revealed that, upon heating, some HK97 capsids exhibit phase transitions at temperatures well below that required for denaturation. We have used cryo-EM to investigate the 60-degree phase transition of a capsid that is fully mature but not crosslinked (as the wildtype mature capsid is). Particles were incubated at 65 degrees for 10 minutes and immediately frozen for observation. As a result of this treatment, they are 12% smaller and have a markedly different structure. This transition is reversible, with a half-life of 2 minutes. These thermally excited capsids appears to represent a previously undetected local minimum in the free energy landscape. They and other thermally excited states appear to capture transitional structures that are short-lived at ambient temperatures. (2b) Regulation of genome packaging. The capsids of double-stranded RNA viruses serve as specialized compartments for the replication and transcription of the viral genomes. We investigate the structural basis of this remarkable phenomenon in the phage phi6 system, which has a tripartite genome. In FY08, we published a paper describing the location of the P2 polymerase inside the viral procapsid. P2 is substoichiometric, occupying only 3 - 10 (depending on the mutant) of 20 potential sites. In FY10 we completed a study using cryo-electron tomography to map the distributions of P2-occupied sites and of the external sites occupied by P4, the packaging ATPase (5). In FY11, we investigated the expansion transformation undergone by the procapsid during RNA packaging. It has been hypothesized that sequential conformational changes in the maturing capsid regulate the order of packaging of the three segments of RNA. To investigate procapsid transformation, we induced expansion in vitro by acidification, heating, and elevated salt concentration. The results identify two structural intermediates between the procapsid and the mature spherical capsid, consistent with this proposal. A paper reporting this study has been submitted for publication.